Hydroxynitrile lyases (HNLs) (EC 4.1.2.10, EC 4.1.2.11, EC 4.1.2.37, EC 4.1.2.39) catalyze the enantioselective cleavage of cyanohydrins to a carbonyl compound (aldehyde or ketone) and hydrocyanic acid.

Reaction Catalyzed by HNL:
In the natural reaction a cyanohydrin (1) is cleaved into a carbonyl compound (aldehyde or ketone) (2) and hydrocyanic acid (3), while a chiral cyanohydrin is produced from the latter ones in industrial approaches.
HNL activity was initially detected in 1908 by Rosenthaler in emulsin from almonds. Representatives of this class of enzyme are primarily found in plants, and occasionally also in insects and bacteria. All known HNLs have been isolated from plants; in nature, the catalyzed reaction serves to ward off aggressors which would feed on them. This release of hydrocyanic acid from cyanohydrins is also termed cyanogenesis. The reverse reaction, i.e. the formation of a chiral cyanohydrin from an aldehyde or a ketone and hydrocyanic acid, which is also catalyzed by HNLs, is used industrially. Chiral cyanohydrins constitute important precursors in the manufacture of β-aminoalcohols and α-hydroxy acids, for example.
The per se highly heterogeneous class of enzymes can be roughly divided into two groups which differ in the presence of the cofactor FAD. The essential FAD molecule appears, however, to have an exclusively structural function; a biochemical significance for the catalyzed reaction has not yet been reported. FAD-containing HNLs have until now only been described for the Rosaceae family—examples of the extensively biochemically characterized representatives are the enzymes from Prunus amygdalus (PaHNL, almond) and Prunus mume (PmHNL, Japanese apricot). The sequences of the enzymes are very similar and all exhibit (R)-selectivity in the formation of chiral cyanohydrins.
The group of non-cofactor-containing HNLs is much more heterogeneous. Examples which can be mentioned are the enzyme from Linum usitatissimum (LuHNL, linseed, (R)-selective) and Sorghum bicolor (SbHNL, millet, (S)-selective). Further, the group includes enzymes from Hevea brasiliensis (HbHNL, rubber tree) and Manihot esculenta (MeHNL, cassava). Although the HNL from linseed has similarities with zinc-dependent alcohol dehydrogenases and that from millet has similarities to carboxypeptidases, the latter enzymes (as well as carboxypeptidases) belong to the α/β hydrolase group and are exclusively (S)-selective. In addition to the common fold motif (see below), the two proteins are also sequentially very similar (77% identity for the amino acids).
In addition to the cited HNLs from cassava and the rubber tree, various enzyme groups such as lipases, esterases, proteases, epoxyhydrolases and dehalogenases belong to the α/β-hydrolase family. All in all, the fold motif consists of mainly parallel β-leaflet strands which are surrounded by α-helices. The active centre is formed by three residues, the so-called catalytic triad (Ollis et al., 1992). The structures of the two HNLs with a α/β hydrolase fold motif (HbHNL, MeHNL) have been resolved and the catalytic residues have been unambiguously identified.
Further differences in the various HNLs concern their substrate spectra and enantioselectivities. The articles by Fechter et al (2004) and Sharma et al (2005) provide an overview of the known enzymes.
Enzymes which are of application on an industrial scale should have a broad substrate spectrum and high enantioselectivity. Furthermore, it is advantageous if the appropriate enzyme can be manufactured in a recombinant manner. An overview of the most important HNLs and their properties is given in Table 1.
TABLE 1Overview of properties of the most industrially important HNLs.RecombinantSubstrateStereo-expressionAldehydeKetoneOriginal organismselectivityhostAliphaticAromaticAliphaticAromaticMiscLiteratureManihot esculentaSE. coli++++Buhler et al.,or MeHNL-“tunnelMethyl ketone2003mutation” W128AHevea brasiliensisSE. coli++++HasslacherP. pastorisMethyl ketoneet al., 1997S. cerevisiaeU.S. Pat. No.6,337,196B1Prunus amygdalusRP. pastoris++++GlycosylatedGlieder et(isoenzyme 5)FADal., 2003cofactorEP 1223220B1Linum usitatissimumRE. coli+−+−Albrecht etP. pastorisal., 1993
Chiral cyanohydrins can be produced with the aid of HNLs in both aqueous systems and in organic solvents such as diisopropylether (DIPE). Frequently, two-phase systems such as DIPE/buffer can be used. The advantage in using organic solvents, in addition to the good solubility of the substrates and products, lies in the suppression of the non-catalyzed chemical reaction of aldehyde/ketone and hydrocyanic acid to racemic cyanohydrin. Since this unwanted reaction is temperature-dependent and only occurs at pHs of more than 5, it can also be prevented by dropping the pH to below 5 and a relatively low reaction temperature (<10° C.). Very recently, moreover, there have been reports of initial tests using HNLs in ionic liquids (Gaisberger et al, 2004). The HNLs in question are used in various preparations: as the dissolved enzyme, lyophilisate, immobilisate on various carrier materials, CLECs (cross-linked enzyme crystals) or CLEAS (cross-linked enzyme aggregates).
U.S. Pat. No. 6,337,196 B1, for example, describes the production of (S)-cyanohydrins with the HNL from Hevea brasiliensis. DE 100 62 306 A1 describes the production of (R)-cyanohydrins with the HNL from Prunus amygdalus. Other specific examples for the use of (R) and (S) cyanohydrins can be found in the articles by Schmidt et al (1999) and Fechter et al (2004).
(S)-selective enzymes, which are currently used industrially, are HNLs from Hevea brasiliensis (HbHNL) and Manihot esculenta (MeHNL); both can readily be produced in microbial hosts and cover a broad substrate spectrum (aliphatic and aromatic aldehydes and ketones, preferably methylketone). The equally (S)-selective enzyme from Sorghum bicolor has until now not been used as it cannot be produced in heterologous hosts.
EP 1 223 220 A1 describes the use of the (R)-selective HNL from Prunus amygdalus (PaHNL, isoenzyme 5). The enzyme can be produced heterologously, but until now only expression in a eukaryotic host (Picha pastoris) has been successful. The equally (R)-selective HNL from linseed (LuHNL) can be expressed heterologously in bacteria, but suffers from the drawback that only aliphatic substrates are accepted and so use on an industrial scale is correspondingly limited.
Thus, there is considerable interest in discovering further (R)-selective HNLs with new enzymatic properties, which in particular are suitable for the transformation of aromatic and aliphatic aldehydes and ketones and in addition can be expressed in good yields in bacterial hosts.